Solexa/illumina Sequencing:
Solexa/illumina also used the technique : Sequencing by synthesis (SBS) with four fluorescentlylabeled nucleotides to sequence short DNA fragments. In compare to 454 platform this Solexa technique labeled deoxynucleoside triphosphate (dNTP) also act as a terminator for polymerization. After each dNTP incorporation, the fluorescent dye is imaged to identify the base and then enzymatically cleaved to allow incorporation of the next nucleotide. Since all four reversible terminator-bound dNTPs (A, C, T, G) are present as single separate molecules, natural competition minimizes incorporation bias.
Step A: Sample/Library preparation
The DNA sample of interest is sheared by focussed acoustic wave with a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-300bp are isolated via gel extraction and amplified using limited cycles of PCR using P5 and P7 primer (Figure 1).
Figure 1a: Amplification of Sequence of interest with P5 and P7 primers
Step B: Cluster Amplification:
The flow cell surface ( Figure: 1b) , a special type of plates for amplification of desired fragments, is coated with single stranded short oligonucleotides that are complemented to the sequences of the ligated adapters during the sample preparation. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell channels and are exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface.
Figure 1b: The Flow cell surface
Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in "cluster station", an automated flow cell processor. Figure 2-7 illustrate stepwise amplification process of cluster.
Figure 2-7: Stepwise illustration of cluster amplification
Step C: Sequencing
A flow cell containing millions of unique clusters is then loaded into the illumina sequencer for automated cycles of extension and imaging.
The first cycle of sequencing starts by adding four labeled reversible terminators, primers, and DNA polymerase and after the first incorporation of a single fluorescent nucleotide, high resolution imaging of the entire flow cell produce the signal. Any signal above background identifies the physical location of a cluster, and the fluorescent emission identifies which of the four bases was incorporated at that position.
This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. Figure 8-13 illustrate stepwise sequencing process.
Figure 8-13: Stepwise illustration of Sequencing process
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